Spermatogenesis in trichosia pubescens (diptera: Sciaridae)

Studies carried out on Sciaridae flies have shown the presence of germ line-limited chromosomes in most of the species. In addition, these studies have revealed that male meiosis is a highly aberrant process (see review in Metz, 1938).

These unusual patterns of chromosome behaviour during the spermatogenesis have been studied in species of the genus Sciara (Bradysia) and Rhynchosciara (Metz, Moses & Hoppe, 1926; review in Metz, 1938; Basile, 1970).

The careful analysis of Sciara spermatocytes allowed Metz and collaborators to trace the history of the chromosomes through meiotic divisions (Metz et al. 1926; Metz, 1933; review in Metz, 1938). These authors concluded that, during the first meiotic division, one haploid complement of somatic chromosomes (S-chromosomes) plus all germ line-limited ones (L-chromosomes) migrate toward one pole of the cell, while the other S-chromosomes go in the opposite direction, being extruded from the cell. The second meiotic division is fairly similar to what is observed in other organisms with the exception of the X chromosome whose chromatids migrate together to the same pole. The chromosome group devoid of X chromosome is extruded from the cell; the other one will constitute the nucleus of the sperm cell. Therefore, in the sciarids, each spermatocyte which undergoes meiosis gives rise to one sperm cell and 2 polar bodies.

In the present work we studied spermatogenesis in Trichosia pubescent. The process in this sciarid is fairly similar, in general features, to that observed in Sciara. However, it was possible to observe some important new aspects regarding the process of chromosome elimination, probably owing to the fixation procedure employed.

T. pubescent were raised as described previously (Amabis, 1974). For the karyotype studies, gonads and brain ganglia from young larvae were dissected in hypotonic solution (75 mM KC1) and kept in a drop of this solution for some 30 min. The tissues were fixed in acetic methanol (1:3) for 30 min and squashed in a drop of 60 % acetic acid plus a drop of lactic-acetic orcein (2 % orcein in a 1:1 acetic acid-lactic acid mixture).

For in vivo analysis of the spermatocytes, testes from old fourth instar larvae and from young pupae were dissected in Cannon’s medium (Cannon, 1964) and, after removing the adhering fat body, they were lightly pressed between a slide and coverslip, in a drop of the medium.

Testes were also fixed for the Feulgen reaction, orcein stain, and autoradiographic preparations. The fixative solution was freshly prepared by mixing 1 vol. of acetic ethanol (1: 3) and 1 vol. of 2 % glutaraldehyde in 0 02 M phosphate buffer. The fixations were carried out for 2-5 min at room temperature. A longer fixation time than this resulted in hardening of the material making separation of the spermatocytes in the squash difficult; a shorter time, on the other hand, did not maintain cell structure. After fixation in this solution, the testes were transferred to a drop of 60 % acetic acid and squashed 2-5 min later. For orcein stain, lactic-acetic orcein was mixed into the acetic acid droplet. The preparations to be used for the Feulgen reaction and for autoradiography were frozen in liquid nitrogen before removing the coverslips.

The autoradiographic studies were conducted on testes from larvae at different stages of the fourth instar and from early pupae. Each larva was injected with 1 μl of a [³H]thymidine solution (New England, sp. act. = 2 mCi/mM; 1 mCi/ml) and sacrificed 20 min or 30-40 h later. Early pupae were injected at stage L_5-6 of the end of the fourth instar (characterized by the size of the eye-spot regions) and sacrificed a few hours after the pupal moult. All slides, after washing in 95 % ethanol, 70 % ethanol, 5 mM acetic acid and in distilled water (each rinse was carried out for 20 min at 4 °C), were coated with Kodak autoradiographic stripping film AR-to and kept in light-proof boxes for 2-3 weeks at 4 °C, before developing according to Kodak instructions.

Analysis of metaphase plates of brain ganglia cells showed that male and female karyotypes have 7 and 8 chromosomes respectively (Fig. 1 A, B). Thus, we concluded that the mechanism of sex determination in this species is the same as described for other Sciaridae; the males are XO and the females XX. Germ-line cells, on the other hand, have 11 chromosomes. Both oocytes and spermatocytes exhibit 4 pairs of somatic chromosomes and 3 chromosomes restricted to germ-line cells (Fig. 1 c).

The nuclei of the spermatocytes from mid fourth instar larvae exhibit, in addition to a light and diffuse chromatin, 3 heterochromatic bodies (Fig. 2 A). When these cells start dividing, the regular chromatin condenses into 8 chromosomes while the heterochromatic bodies undergo a slight decondensation resulting in 3 chromosomes (Fig. 2B, c). The latter could be characterized as L-chromosomes due to their difference in size when compared to the S-chromosomes (Fig. 1c). Thus, one can conclude that the 3 heterochromatic bodies present in the interphase spermatocyte nuclei represent the 3 L-chromosomes in a highly condensed state. We have found in our stocks a few males having 4 L-chromosomes, instead of 2 large and 1 short elements. These males showed 2 large and 2 short L-chromosomes. Variation in the number of L-chromosomes within a single species was also found in Sciara coprophila (Rieffel & Crouse, 1966).

Exploratory studies carried out on a variety of larval and pupal stages showed that meiosis in T. pubescens takes place in the early pupal stage. In most of the testes prepared at this specific stage, we could observe cells in different phases of the meiotic cycle, showing that meiosis is asynchronous even within a single testis. Analysis of the living and fixed testes preparations have shown that the cells within the testis are associated in clusters of some fifteen spermatocytes (Figs. 3, 4). These clusters (rosettes) have a globular appearance and all cells belonging to a single cluster are fairly similar with regard to their phase in the meiotic cycle (Figs. 3, 4).

The alterations in spermatocyte structure which occur during the first hours after pupal moult can easily be ordered in a logical sequence of events which is presented as follows (Fig. 5).

The first morphological change in spermatocytes of early pupae is the protrusion of a narrow neck of cytoplasm with a small cap at the apex (Figs. 2B, 4, 5 A, B). This cellular projection (bud) elongates during the meiotic cycle becoming a fingerlike structure by the end of the first division (Figs. 3, 5C-1, 7A). The cytoplasmic protrusion always takes place at the pole of the cell facing the centre of the rosette (Figs. 3, 4). This process starts simultaneously with or a little before the beginning of condensation of the S-chromosomes (Figs. 2B, 4, 5 A).

During condensation of the S-chromosomes, the L-chromosomes undergo a decompacting process passing from a highly condensed state, characteristic of interphase, to a state of condensation similar to that exhibited by the meiotic S-chromosomes (Figs. 2C, 4).

Meiotic prophase in males of T. pubescens is typically achiasmatic; the synapsis is absent and the chromosomes are always univalent and diploid in number (Figs. 4, 5 A).

When the chromosomes attain a highly condensed state, the boundary between the nucleus and the cytoplasm disappears. The chromosomes, however, do not move to the equatorial region of the cell to form a metaphase plate. From a dispersed state in the centre of the cell, they separate into 2 groups which move toward opposite poles (Figs. 4, 6). Of the 11 chromosomes, 4 migrate budward while the other 7 move in the opposite direction. The migration of the 7 chromosomes to the pole opposite to the bud is not a synchronized process. For instance, in Fig. 4, one can see 2 cells in which i chromosome has already migrated while the others remain dispersed in the centre of the cell. In addition, all chromosomes migrating to the pole away from the bud have a typical anaphase configuration (V-shaped), suggesting that they are being pulled by centromeric regions which have a median or submedian location in these chromosomes (Fig. 6). When cells at this specific stage are carefully examined it is seen that the 4 chromosomes moving toward the bud never exhibit a V-shape as would be expected if they were being pulled by the centromeric regions (Fig. 6); these 4 chromosomes do not exhibit any consistent form during migration. The other 7 chromosomes maintain their V-shape until the 4 chromosomes at the bud ‘pole’ penetrate into the narrow cytoplasmic projection. Inside the bud, the 4 chromosomes clump together forming a heterochromatic mass which continues to migrate toward the distal portion of the cell protrusion (Figs. 5 c, D, 7A). After penetration of the 4 chromosomes into the bud, those chromosomes at the opposite pole lose their V-shape, decondense, and a nucleus which occupies the centre of the cell body is reconstituted (Fig. 5 c).

The second meiotic division is fairly similar to the ordinary type observed in other organisms except that both chromatids of one chromosome (probably the X) migrate precociously toward the same pole of the cell (Figs. 5E, 7B). The chromosomes in the nucleus start to condense (Figs. 5D, 7A). The limit between the nucleus and the cytoplasm disappears and the chromosomes move to the equatorial region of the cell constituting a typical metaphase plate (Figs. 5E, 7B). Thus, at metaphase II, the situation is the following: 6 chromosomes remain in the centre of the cell forming a flat equatorial plate, the seventh is regularly found moving toward or is at the pole away from the bud. The chromosomes at the equatorial plate split longitudinally sending daughter halves to opposite poles. All chromosomes show, during migration, a V-shape, as would be expected if they were being pulled by their centromeres (Figs. 5F, 8A, B).

At the first meiotic division we could observe, with phase microscopy, some oriented material running from the pole opposite the bud up to the equatorial region of the cell (Fig. 9). At the second division, on the other hand, it is easy to see oriented material running from the proximal portion of the cytoplasmic protrusion up to the opposite pole (Figs. 5E, 7o, 8B, 1O). These findings suggest that a typical bipolar spindle is present during the second meiotic division and that one of the poles of the spindle is located inside the cytoplasmic protrusion. Actually, the 6 chromosomes moving toward the pole of the cell facing the centre of the rosette penetrate inside the bud without losing their V-shape (Fig. 8 A, B). When these chromosomes reach halfway between the origin of the bud and the first extruded chromosome group they stop moving and start to decondense (Figs. 5G-1, 10, IIA, B). Both chromosome groups which segregate at the second meiotic division decondense simultaneously forming 2 interphase nuclei: one in the centre of the cell body and the other in the median region of the bud (Figs. 5G, H, IIA).

The chromosomes eliminated at the first division remain clumped together in a highly condensed state (Figs. 5 H, 1, 10, 11 A, B). Thus, just after the end of the second meiotic division, each spermatocyte presents the following situation: a set of 4 doublestranded chromosomes remains, in a highly heterochromatic state, in the distal portion of the cytoplasmic protrusion; 6 single-stranded chromosomes, within a typical interphase nucleus, occupy the median region of the cytoplasmic protrusion; and a set of 8 single-stranded chromosomes constitutes the typical interphase nucleus that remains in the central region of the cell body. The latter chromosome group will constitute the nucleus of the sperm cell.

After telophase II, the cell lengthens along the axis oriented with the radius of the rosette, determined by the cell-division poles (Figs. 51,11 B). The nucleus containing the 8 chromosomes remains in the proximal portion of the spermatid and the other 2 chromosome groups will occupy its more distal portion, being extruded during the formation of the spermatozoon tail

In order to verify when premeiotic DNA synthesis takes place, larvae at different stages, at the end of the fourth instar, and early pupae, were injected with a solution of [³H]thymidine and sacrificed 20 min later and their testes were used for autoradiographic preparations. Analysis of these autoradiograms showed that spermatocytes of pre-pupae (larvae after migration of the eye-spot region) and pupae do not incorporate [³H]thymidine. The spermatocyte nuclei of younger larvae, however, became intensely labelled after a [³H]thymidine pulse. In these spermatocytes 3 kinds of labelling pattern were observed: diffuse labelling over all the nucleus; labelling only over the diffuse chromatin, with the L-chromosomes unlabelled; and labelling restricted to the L-chromosomes (Fig. 12 A).

Prior to the pre-pupal stage, i.e. by the end of premeiotic DNA synthesis, only a few nuclei incorporated the labelled precursor. In these larvae, most labelled nuclei presented only the L-chromosomes labelled. These results show that replication of the L- and S-chromosomes are asynchronous, with the L-chromosomes being later in replicating.

When larvae were injected before migration of the eye-spot region and sacrificed 30-40 h later, at the early pupal stage, different kinds of labelling patterns could be observed in the meiotic spermatocytes. Besides a high number of unlabelled cells, we could see labelled spermatocytes characteristically at the end of the first and the second meiotic divisions. Two labelling patterns were observed in the cells at the end of the first meiotic division: spermatocytes with label restricted to the chromosome set at the pole opposite the bud (type I) and spermatocytes with both chromosome groups labelled (type II) (Fig. 13). Spermatocytes characteristically at the end of the second meiotic division displayed labelling over the 3 chromosome groups (type III) as well as label restricted to the 2 sets that segregated at this division (type IV) (Fig. 13: Fig. 13 c shows an intermediate pattern between types III and IV).

If we assume that the L-chromosomes of T. pubescens are late in replicating and that premeiotic DNA synthesis ends just before the pre-pupal stage, we can interpret the results obtained with the larvae injected with [³H]thymidine 30-40 h before meiotic division as follows. By the time of [³H]thymidine injection, some spermatocytes had already finished the premeiotic DNA synthesis; others were at the end of this process, replicating just the L-chromosomes, and still others were at the beginning or middle of S-phase, with the S-chromosomes being replicated. The first class of cells would appear unlabelled in autoradiograms prepared 30-40 h after precursor injection. The second class would display patterns I and IV since the first-eliminated chromosomal set does not contain L-chromosomes. The third cell class would present patterns II and III since all chromosomal sets formed during male meiosis have S-chromosomes.

In T. pubescens, as has been observed in other Sciaridae (Rieffel & Crouse, 1966; Basile, Casartelli & Benozzati, 1974), the germ-line limited chromosomes are typically heterochromatic: during interphase, they stain darker than the S-chromosomes and they undergo DNA replication out of phase with the rest of the cellular DNA, probably at a later stage of the cell cycle. On the other hand, Rieffel & Crouse (1966) suggested that the L-chromosomes of Sciara coprophila are active early in embryogenesis when, during interphase, they become very long, assuming a diffuse aspect. As pointed out by these authors, ‘it seems unlikely that the germ cells would retain the chromosomes so regularly if they had no function in gonad development’. We have observed that the L-chromosomes of T. pubescens spermatocytes become decondensed and indistinguishable from the S-chromosomes during the interphase between the meiotic divisions and just after the end of the second meiotic division. This could mean that DNA sequences present in the L-chromosomes are transcribed during these stages. This hypothesis is comparable to what happens in the spermatogenesis of Drosophila. In the latter organisms, the Y chromosomal heterochromatin is required for a certain process in the spermatogenesis (Hess & Meyer, 1963). The Y chromosome of Drosophila spermatocytes decondenses and becomes active at this time (see review in Hess, 1973).

Our interpretation of the spermatogenesis figures observed in the preparations of T. pubescens testes are summarized in Fig. 5.

Comparison of these results with those obtained in other Sciaridae (Metz et al. 1926; review in Metz, 1938; Basile, 1970) leads to the conclusion that chromosomal behaviour during spermatogenesis of different sciarid species is fairly similar, if not identical. Our findings differ from those described by Metz and coworkers (Metz, Moses & Hoppe, 1926) in the mechanism of bud formation and the fate of the eliminated chromosomes.

In describing the end of the first meiotic division in the Sciara male, those authors said (Metz et al. 1926, p. 248): ‘After the four chromosomes have come together near the periphery of the cell as described above, an évagination occurs and a narrow neck of protoplasm is seen to protrude with the chromosomes in its apex.’ This and other descriptions of bud formation (Metz, 1933, 1938) suggest that in Sciara this event is similar to the formation of a polar body in an egg. Our results in T. pubescens, however, show that bud formation starts prior to the first meiotic division, being markedly at difference with what was described in Sciara. The differences between these species could reflect variations in a common process of chromosome elimination. However, when we used only acetic ethanol to fix the testes, the buds were scarcely visible in T. pubescens spermatocytes, the meiotic figures found in these preparations being quite similar to those so far described in different species of Sciaridae (Metz et al. 1926; Metz, 1933; Basile, 1970; Crouse, Brown & Mumford, 1971). Taking into account these findings, we believe that differences observed in the mechanism of chromosome elimination between T. pubescens and the other sciarids so far studied are due to the fixation procedures used. Such a conclusion, however, may only be attained if other species are studied with the fixation procedure used here.

Another conflicting point between our results in T. pubescens and those described by Metz et al. (1926) in Sciara concerns the fate of the chromosomal set eliminated at the first meiotic division. In Sciara, these authors described the eliminated chromosomes as surrounded by little, if any, cytoplasm separate from the cell body, and that they formed an interphase nucleus with a more or less net-like internal structure. We have shown that in T. pubescens the first-eliminated set of chromosomes remains inside the bud, in a highly heterochromatic state, up to the end of spermatogenesis. The description in Sciara, however, completely agrees with what we observed for the chromosomal set eliminated at the second meiotic division, except that, in T. pubescens, the bud nuclei remain associated with the cell body until later in the spermiogenetic process. Again, these variations between Sciara and Trichosia could reflect a difference in the basic process or could be a misinterpretation of the figures observed in the former. In addition, the formation of a second bud at the end of the second meiotic division was described in Sciara while, in Trichosia, both chromosomal sets are eliminated inside a single cytoplasmic protrusion.

Regarding chromosomal migration during the meiotic divisions of T. pubescens spermatocytes, one can conclude that 2 different mechanisms of chromosomal movement are involved. The migration of the chromosomes toward the bud cannot be explained by the standard theories.

We have observed that the chromosomes migrating to both opposite poles during the second meiotic division and those migrating toward the pole opposite to the bud during the first division, displayed a V-shape. The V-shape exhibited by metacentric or submetacentric chromosomes during anaphase is considered to result from the motion forces acting at their kinetochore (see review in Nicklas, 1971). Thus, the movement of these chromosomes could be explained by the association of their median kinetochore with the spindle fibres which would produce the forces that lead them polewards. On the other hand, the chromosomes which move budward, during the first meiotic division, never exhibit a V-shape as would be expected if they were being pulled by forces acting at their kinetochores. This fact suggests that a motion mechanism, other than median kinetochore association to spindle fibres, is responsible for this unorthodox chromosomal behaviour. This conclusion is supported by the fact that, during the first meiotic division, oriented material is only seen running from the bud-opposite pole up to the equatorial region of the cell, while a typical bipolar spindle is observed during the second division. Similar observations were made by Metz and coworkers (Metz et al. 1926; Metz, 1933) in Sciara.

The other mechanism of chromosomal motion observed in some lower eukaryotes (see review in Kubai, 1975) depends upon chromosome attachment to nuclear membrane and is determined by membrane growth. We do not believe that such a mechanism is responsible for the chromosome motion toward the bud during the first meiotic division of Sciaridae spermatocytes. Other kinds of forces, such as cytoplasmic movement could drag these chromosomes toward the bud pole. If this is true, the pole kinetochore association exhibited by the chromosomes settled in the region away from the bud, would reflect more an anchor state than a motion process. The association of those chromosomes to that pole would prevent them from being dragged toward the bud. This hypothesis leads us to postulate that the fate of a chromosome during the first meiotic division (that is, being eliminated or remaining in the cell body) depends exclusively on its kinetochore capability to attach to the unique spindle pole.

The studies carried out in Sciara (see review in Metz, 1938) lead to the conclusion that the 4 chromosomes eliminated during the first division of the spermatocytes all belong to the paternal S-chromosomal set. Thus, an S-chromosome inherited from the father has an imprint which permits its recognition at the first meiotic spermatocyte division (Crouse, 1960). Taking into account the hypothesis that the maintenance of specific chromosomes in the spermatocyte body during the first meiotic division is dependent upon its attachment to the spindle fibres, the imprint of the paternal S-chromosomes could be specific alterations in their kinetochores which would prevent them from associating specifically with the monopolar spindle fibres.

The chromosomal elimination mechanism proposed is quite attractive although speculative. We believe that electron-microscopic studies of these cells will supply information on the structure of the monopolar spindle, giving additional basis for elucidation of the chromosomal elimination mechanism in Sciaridae.

We should like to thank Mr D. Barros for his help during the experiments and to Dr L. C. G. SimSes and Ms Tamara J. Lister for critical reading of the manuscript. This work was supported by FAPESP (77/1034) and CNPq-PIG (SIP-04/001).